Solution phase isoelectric fractionation in the multi-compartment electrolyser: a divide and conquer strategy for the analysis of complex proteomes.

Sample complexity frequently interferes with the analysis of low-abundance proteins by two-dimensional gel electrophoresis (2DGE). Ideally, high abundance proteins should be removed, allowing low-abundance proteins to be applied at much higher concentrations than is possible with the unfractionated sample. One approach is to partition the sample in a manner that segregates the bulk of extraneous proteins from the protein(s) of interest. Solution phase isoelectric focusing in the multi-compartment electrolyser generates fractions of discrete isoelectric point (pI) intervals allowing isolated narrow segments of a proteome to be analysed individually by 2DGE. It is particularly useful for the isolation of low-abundance proteins of extremely basic or acidic pI.     

Purification of plasmid DNA using a multicompartment electrolyser separated by ultrafilter membranes

A simple, scalable method for purification of plasmid DNA is described. Plasmid DNA was released from Escherichia coli JM109 by lysis (1% SDS, 0.2 M NaOH). Then a neutralization solution (3 M sodium acetate buffer, pH 4.8) was added to precipitate genomic DNA and protein. After the clarification of the lysate, the supernatant was placed in a multicompartment electrolyser separated by ultrafilter membranes to remove the remaining contamination (RNA, genomic DNA and protein). A recovery of 75%±2% of total plasmid DNA was obtained after 60 min electrophoresis with a field strength of 8 V cm^–1 using cells at 30 g l^–1 (quantified by dry cell weight). Genomic DNA, RNA and protein were undetectable in the purified plasmid DNA solution.     

Spatial Localization of Synapses Required for Supralinear Summation of Action Potentials and EPSPs

Although the supralinear summation of synchronizing excitatory postsynaptic potentials (EPSPs) and backpropagating action potentials (APs) is important for spike-timing-dependent synaptic plasticity (STDP), the spatial conditions of the amplification in the divergent dendritic structure have yet to be analyzed. In the present study, we simulated the coincidence of APs with EPSPs at randomly determined synaptic sites of a morphologically reconstructed hippocampal CA1 pyramidal model neuron and clarified the spatial condition of the amplifying synapses. In the case of uniform conductance inputs, the amplifying synapses were localized in the middle apical dendrites and distal basal dendrites with small diameters, and the ratio of synapses was unexpectedly small: 8-16% in both apical and basal dendrites. This was because the appearance of strong amplification requires the coincidence of both APs of 3-30 mV and EPSPs of over 6 mV, both of which depend on the dendritic location of synaptic sites. We found that the localization of amplifying synapses depends on A-type K+ channel distribution because backpropagating APs depend on the A-type K+ channel distribution, and that the localizations of amplifying synapses were similar within a range of physiological synaptic conductances. We also quantified the spread of membrane amplification in dendrites, indicating that the neighboring synapses can also show the amplification. These findings allowed us to computationally illustrate the spatial localization of synapses for supralinear summation of APs and EPSPs within thin dendritic branches where patch clamp experiments cannot be easily conducted.     

Scaling-down biopharmaceutical production processes via a single multi-compartment bioreactor (SMCB)

Biopharmaceutical production processes often use mammalian cells in bioreactors larger than 10,000 L, where gradients of shear stress, substrate, dissolved oxygen and carbon dioxide, and pH are likely to occur. As former tissue cells, producer cell lines such as Chinese hamster ovary (CHO) cells sensitively respond to these mixing heterogeneities, resulting in related scenarios being mimicked in scale-down reactors. However, commonly applied multi-compartment approaches comprising multiple reactors impose a biasing shear stress caused by pumping. The latter can be prevented using the single multi-compartment bioreactor (SMCB) presented here. The exchange area provided by a disc mounted between the upper and lower compartments in a stirred bioreactor was found to be an essential design parameter. Mimicking the mixing power input at a large scale on a small scale allowed the installation of similar mixing times in the SMCB. The particularities of the disc geometry may also be considered, finally leading to a converged decision tree. The work flow identifies a sharply contoured operational field comprising disc designs and power input to install the same mixing times on a large scale in the SMCB without the additional shear stress caused by pumping. The design principle holds true for both nongassed and gassed systems.     

Reassessing Models of Hepatic Extraction

The aim of this investigation is to compare different mathematical models of the liver in the context of in vitro-in vivo correlation. We reanalyze drugs from the Houston reviews [1, 2], and compare the mathematical models. For the well-stirred model, a particular form of the distributed tubes model, and the dispersion model, fits are done to in vitro and in vivo intrinsic clearance data from microsomal and hepatocyte experiments. The distributed and dispersion models have decreased residuals as compared to the well-stirred model, but neither is to be clearly preferred over theother. It seems likely that drug-specific factors have a major impact on the quality of IVIVC correlations. While new experiments are needed to validate IVIVC models, our results indicate that improved correlation of in vitroand in vivo data is possible for high clearance drugs by using either a dispersion or distributed tube model rather than a well-stirred model.